The present invention relates generally to methods of regulating the starch composition of plants. In particular, the present invention relates to novel UDP-glucose dehydrogenase (UDPGdH) genes, mutant and variant forms of the genes, and the use of such UDPGdH genes to produce plants with altered starch content due to modified UDPGdH activity.
Polysaccharides produced by plants are useful for a variety of home and industrial applications. Plant gums and starches, for example, are used extensively in food and pharmaceutical industries, due to their emulsifying, stabilizing, thickening, and gel-forming properties (see, for example, Sandford and Baird, in The Polysaccharides (Aspinall, ed.), 2:411 (Academic Press 1983); Azczesniak, in Gums and Stabilisers for the Food Industry (Phillips et al., eds), 3:311 (Elsevier Applied Science Publishers 1986)). It would therefore be beneficial to alter plant polysaccharide synthesis to improve the quantities of polysaccharides or to provide new types of polysaccharides. Modifications of polysaccharide synthesis also can lead to improved plants. For example, increasing the amount of cell wall polysaccharide during development would improve resistance of developing seeds to pathogens and insects resulting in improved crop yields.
UDP-glucose is a nucleotide sugar that occupies a central position in plant metabolic pathways, including the synthesis of polysaccharides. UDP-glucose serves both as a precursor in sucrose synthesis and in the formation of those sugar nucleotides required for the synthesis of cell wall components. for example, UDP-glucose metabolism leads to amino sugars, cellulose, sucrose, fructans, and other non-cellulosic polysaccharides. In addition, UDP-glucose can substitute for ADP-glucose as a starch synthase substrate, at least in the waxy maize phenotype.
UDP-glucose dehydrogenase (UDPGdH) [EC 1.1.1.22] is an NAD+-linked, four-electron transferring oxidoreductase that converts UDP-D-glucose (UDP-G) to UDP-D-glucuronic acid (UDP-GA) by two oxidation reactions in which UDP-6-aldehydo-D-glucose is an intermediate (Hempel et al., Protein Science 3:1074, 1994). UDP-GA is a precursor for sugar nucleotides, which are required for the biosynthesis of various components of hemicellulose, including arabinans, arabinogalactans, glucuronoarabinoxylans, rhamnogalacturonans, xylans, and xyloglucans. Evidence indicates that UDPGdH catalyzes the rate-limiting step in the synthesis of these cell wall precursors (Witt, Journal of Plant Physiology 140:276, 1992). UDPGdH is also centrally involved in the production of a variety of exopolysaccharide gums including xanthan gum and a variety of non-commercial gums produced by Streptococci (Ashtaputre and Shah, Current Microbiology 31:234, 1995); Lin et al., Biochemical and Biophysical Research Communications 207:223, 1995).
Since it is difficult to control the quantity and composition of hemicellulosic substances, there are problems with extraction of other useful plant-derived fibers and substances. Further, many naturally-occurring hemicellulosic substances and other polysaccharides derived from UDPGdH products are not useful commercial products due to their insufficient quantity and varying composition. Accordingly, there is a need for a means to improve and regulate the production of plant polysaccharides.
The present invention exploits novel genes that are highly and specifically expressed in developing plants to alter the regulation of UDPGdH activity, thus providing control of plant polysaccharide synthesis.
The present invention provides nucleic acid molecules that encode plant UDP-glucose dehydrogenase (UDPGdH), as well as modified UDPGdH proteins. More specifically, one aspect of the present invention provides isolated nucleic acid molecules encoding maize UDPGdH. Such nucleic acid molecules comprise a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or a variant thereof. Within one embodiment, the nucleotide sequence is either SEQ ID NO:1 or SEQ ID NO:3.
A second aspect of the present invention provides modified nucleic acid molecules encoding mutant forms of UDPGdH in which the catalytic cysteine 272 residue has been replaced with another amino acid residue. These mutant enzymes predominantly catalyze the conversion of UDP-D-glucose to UDP-6-aldehydo-D-glucose as the end product.
Also provided by the present invention are polypeptides encoded by such nucleotide sequences, vectors comprising such nucleotide sequences, and host cells that contain these vectors. Suitable vectors include expression vectors, such as a binary Agrobacterium tumefaciens plasmid vector. Suitable expression vectors can contain a seed-specific, tissue-specific, cell type-specific, or a plastid-specific promoter. Representative examples of suitable host cells include plant cells from maize, sorghum, wheat, rice, barley, oats, and potato. Preferred plant cells are maize cells.
Yet other aspects of the present invention include methods for producing a plant that expresses UDPGdH, comprising the steps of (a) introducing a vector as described above into an embryogenic plant cell, wherein the vector contains a UDPGdH gene in an expressible form, and (b) producing a plant from the embryogenic plant cell, wherein the plant expresses the UDPGdH gene. A nucleic acid molecule encoding UDPGdH also can be inserted in an expression vector to produce UDPGdH protein in host cells, such as bacterial, yeast, mammalian, insect or plant cells.
In one embodiment, over-expression of the UDPGdH gene in plant cells is used to increase polysaccharide synthesis. In other embodiments, expression in plant endosperm amyloplasts of UDPGdH enzyme activity is used to produce carboxylated starch. In additional embodiments, expression of exogenous UDPGdH enzyme activity by transgenic plants increases stem strength or stalk strength, and enhances resistance to insects and pathogens.
The present invention further provides methods for inhibiting endogenous UDPGdH activity in plant cells using UDPGdF nucleotide sequences. Plants comprising cells having diminished UDPGdH activity have enhanced value as an industrial source for starch and as animal feed. Such enzyme inhibition can be achieved, for example, by co-suppression. Alternatively, nucleotide sequences encoding at least a portion of the UDPGdH gene are inserted in the anti-sense direction into expression vectors to inhibit the expression of the UDPGdH gene in plant cells.
The present invention also provides methods of producing a plant that produces polysaccharides comprising 6-aldehydo-D-glucose moieties, and transgenic plants and plant cells that contain the nucleic acid molecules. or vectors, described herein. A method for producing polysaccharide that contains 6-aldehydo-D-glucose moieties, such as aldehydic starch, comprises the steps of (a) introducing into a plant cell an expression vector that comprises a mutant UDPGdH gene, and (b) growing the plant cell under conditions wherein the mutant UDPGdH gene is expressed. The present invention further includes polysaccharides comprising 6-aldehydo-D-glucose moieties, produced by transgenic plant cells.
The present invention also provides isolated nucleic acid molecules comprising a nucleotide sequence that encodes a polypeptide having UDP-glucose dehydrogenase (UDPGdH) activity, wherein the nucleic acid molecule is selected from the group consisting of (a) a polynucleotide that encodes an amino acid sequence having at least 80% identity to the amino acid sequence of either SEQ ID NO:2 or SEQ ID NO:4, with the proviso that the amino acid sequence is not encoded by the soybean UDPGdH gene having the nucleotide sequence of GenBank accession No. U53418, (b) a polynucleotide of at least 30 nucleotides in length which selectively hybridizes under stringent conditions to a nucleic acid molecule having the nucleotide sequence of either SEQ ID NO:1 or SEQ ID NO:3, (c) a polynucleotide that is complementary to polynucleotide (a) or (b), and (d) a polynucleotide comprising at least 30 contiguous nucleotides from polynucleotide (a), (b), or (c). A suitable polynucleotide (a) encodes a polypeptide that elicits the production of an antibody which is specifically reactive to a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. The present invention also contemplates expression cassettes that comprise a promoter operably linked to polynucleotides (a), (b), (c) or (d), in either the sense or anti-sense orientation. The present invention further provides recombinant host cells that comprise such expression cassettes. The present invention also includes isolated proteins comprising a polypeptide of at least ten contiguous amino acids encoded by such nucleic acid molecules.
The present invention also provides isolated nucleic acid molecules that encode a polypeptide having UDP-glucose dehydrogenase (UDPGdH) activity, and that can hybridize under stringent conditions with a nucleic acid molecule having either SEQ ID NO:1 or SEQ ID NO:3.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are identified below and are incorporated by reference in their entirety.